Prothrombinase is the coagulation complex composed of the serine protease factor Xa, the protein cofactor factor Va, phospholipid or cellular surface and calcium ions. This supramolecular complex catalyses the proteolytic activation of the zymogen prothrombin to the active protease alpha-thrombin. Highly specific, active site-directed fluorescent probes have been developed to some of the prothrombinase constituents. The approaches outlined in this proposal will utilize these available fluorophores to study the dynamic aspects of the prothrombinase complex by stopped-flow kinetics. Factor Xa, covalently modified at the active site with dansyl glutamylglycinylarginyl chloromethylketone, (DEGRCK), will be used to study the kinetic mechanism of prothrombinase assembly by fluorescence stopped- flow. The fluorescent alpha-thrombin inhibitor dansylarginine-N- (3-ethyl-1,5-pentanediyl)amide (DAPA), will be used in stopped- flow experiments to study prothrombinase assembly in the presence of substrate and to evaluate the early events in the catalytic cycle of the enzyme. Stopped-flow studies of prothrombinase assembly in the presence or absence of substrate will be conducted using factor Va and other factor V-derived peptides in order to better define regions of the cofactor that participate in the assembly and function of the complex. Studies are also proposed to determine the kinetic parameters for the inactivation of factor Va by activated protein C in the presence and absence of protein S and to explore the competition between factor Xa and activated protein C by kinetic and equilibrium approaches. The development of a blood clot is a highly amplified response that is confined to the site of vascular injury. The proposed studies will provide information relevant to the assembly, function and regulation of prothrombinase. This information will result in and increased understanding of clot formation under the dynamic conditions of blood flow.